TABLE 2.

DNA methylation studies in nasal epithelial cells

Study population	Ethnicity‐origin	Age [average (range), or: mean ± SD/SEM]	Gender (male%)	Hospital OR population‐based cohort	Asthma/AR diagnosis	Sampling method	Methylation measurement method	Important findings	Reference
35 asthmatic children‐ non controls	Caucasian	8.9	77%	Population	Physiciandiagnosis of asthma; reported wheezing symptoms in the previous 12 months; chesttightness and/or use of bronchodilators in the last 12 months	Nasal cell brushing‐Inferior turbinate	PCR‐pyrosequencing on bisulfite‐treated DNA	↓ DNA methylation of the IL‐6 and iNOS promoters ‐ ↑ FENO	Baccarelli et al. (2012) 40
								Alu and LINE‐1 methylation showed no associations with FENO and FEV1
Discovery: N = 15 asthmatic	33.3% Caucasian	13.4 (7.4–18.0)	73.3%	Hospital	Respiratory symptom score (based on frequency of wheeze, cough, shortness of breath, and chest tightness) and the age‐specific asthma control Test™ scorewas collected	Nasal mucosa brushing‐ epithelial cells (CytoSoft Brush)	Targeted PCR‐pyrosequencing on differentially expressed genes between good and poor responders	Altered DNA methylation at VNN1 promoter‐ VNN1 as a biomarker for corticosteroid treatment	Xiao et al. (2015) 41
Replication N = 25 asthmatics	8% Caucasian	8.1 (5.0–15.1)	64%	Hospital
N = 20 exacerbating asthmatics‐	20% White 70% Black 10% biracial	8.0 (6.0–15.0)	80%	Hospital	Respiratory symptom score (based on frequency of wheeze, cough, shortness of breath, and chest tightness) and the age‐specific asthma control Test™ score was collected	Nasal mucosal brushing‐ epithelial cells (CytoSoft Brush)	Llumina Infinium HumanMethylation450 BeadChip (Illumina)	182 CpGs with common SNPs overlap (LDHC, DNHD1 and PRRC1) 127 CpGs without SNP co‐localization (GALNTL4, SRD5A3, MED12L‐GPR87 locus and CARD14) ↓ methylation of OTX2 gene after treatment	Zhang et al. (2017) 42
N = 13 more exacerbating asthmatics	15%white77%black8%biracial	6 (5.0–8.0)	46%	Hospital
Population 1 N = 10 asthmatics (atopic) N = 7 healthy controls	Multiracial	23 (±2.4); 26 (±5.9)	Asthmatics 40%; Healthy 14.3%	Population	Individuals with a history of asthma. Determination of additional clinical characteristics: Airway hyperreactivity, lung function and atopic status via methacholine challenge, spirometry and allergen skin test respectively	Nasal Scrapings of the medial inferior surface of the inferior turbinate after local vasoconstriction and anesthesia	Pyrosequencing global DNA methylation (Alu elements)	↑ global methylation in response to virus infection	McErlaean et al. (2014) 43
Population 2 N = 6 asthmatics (atopic) N = 6 healthy controls		20 (0.9); 31 (9.8	Asthmatics 33.4% Healthy 84%				Infinium human methylation 450 K bead chip microarray (Illumina)	SNORA12 different methylation between atopic asthmatic and healthy subjects
N = 10 asthmatic children N = 10 healthy children (ALLIANCE study)	N/A	11.6 (8–15) – full term birth	50%	N/A	Asthma diagnosis based on the 2014 global initiative for asthma (GINA) guidelines	Nasal brushing (3 nm IDB‐G brush)	Infinium human methylation 450 K bead chip microarray (Illumina)	HRVI mediated methylation changes to: AGPAT1, BAT3, NEU1, ANAPC11, MGST3, CCT6A, MICB, SMN1, LOC442454, KLHL8, SLC16A3, GP1BA, DNAJC7, VDAC2, FBXO7, TP53I3	Pech et al. (2018) 44
							Confirmation study: Pyrosequencing
Discovery N = 35 asthma‐controls sibling pairs (exposure Sibling Study)	African‐American	11.0 (9.0–14.0)	54%	Mixed	Asthma diagnosis was obtained from the parental report, and confirmed via electronic medical record. (Research electronic data capture‐ REDCap software characterized asthma onset, diagnosis, symptoms, severity, quality of life, medication, environmental exposures, social histories and residential address for the first year of life and for the past 5 years.	Nasal epithelial cell or nasal mucosa sampling with a CytoSoft Brush	Infinium HumanMethylation450 BeadChips (N = 12); Validation via Pyrosequencing (N = 35)	↓ DNA methylation at TET 1 promoter	Somineni et al. (2016) 45
Replication N = 158 asthmatics N = 28 non asthmatics		12.0 (8.0–15.0)	52%		Asthma diagnosis according to American Thoracic Society criteria	Saliva	Replication via Pyrosequencing (N = 186)
Discovery N = 54 asthma‐ controls sibling pairs (exposure Sibling Study)	African‐American	12.01 (5–18) Asthmatics; 11.35 (5–18) controls	Asthmatics 63%; Non‐asthmatics 43%	Mixed	Asthma diagnosis was obtained from the parental report, and confirmed via electronic medical record. (Research electronic data capture‐ REDCap software characterized asthma onset, diagnosis, symptoms, severity, quality of life, medication, environmental exposures, social histories and residential address for the first year of life and for the past 5 years:	Nasal epithelial cell or nasal mucosa sampling with a CytoSoft Brush	Infinium HumanMethylation450 BeadChips (N = 58); Validation pyrosequencing (N = 108)	6 CpGs associated with asthma(no statistical significance) ↓3 non‐SNP CpGs replicated in independent cohots associated to TET1, OR2B11 and NLRP3	Zhang et al. (2018) 46
Discovery study (inner city Consortium) N = 36 cases with persistent asthma N = 36 healthy controls without atopy or asthma (shared controls)	African American 91.7%; Hispanic Latino 8.3%	11 (10–12)	Asthmatics 52.8% Controls 47.2%	Population	(1) physician diagnosis; (2) persistent or uncontrolled disease defined by the National asthma Education and Prevention Program; (3) physiologic evidence of asthma (FEV1 < 85% or FEV1/FVC ratio<85%; and bronchodilator responsiveness (≥12%) or PC20 < 8 mg/ml of methacholine); (4) Positive skin prick test to at least one of a panel of indoor aeroallergensAtopy assessed by positive skin prick testing for multiple allergens or RAST/Phadiatop tests.	Nasal epithelial cells brushing from the posterior portion of the inferior turbinate	Illumina's Infinium human methylation 450k BeadChip	119 genome‐wide significant DMRs associated with 118 unique genes 118 single CpG motifs (DMPs) associated with 107 unique genes: Some gene associated to asthma: ALOX15, CAPN14, HNMT, POSTN Extracellular matrix: COL16A1, COL5A2, COL5A3, ELN, HAS3, MMP14, Immunity: IFNGR2, HLKA‐DPA1, LAG3, NFIL3, PRF1, TNFSF13 Cell adhesion CTNND1, EPPK1, GJA4 epigenetic regulation ATXN7L1, H1F0, HIST1H1D, METTL1 Airway obstruction GABRG3, Obesity C1QTNF1, GPC4 Autophagy AMBRA1	Yang et al. (2017) 47
Validation 1 N = 30 asthmatics N = 36 shared controls from discovery study	African American	11 (10–12)	N/A				Pyrosequencing ‐targeted genes
Validation 2 N = 12 asthmatics and N = 12 controls without asthma.	Caucasian	24–74	N/A		(1) FEV1 > 55% at visit 1; (2) positive methacholine challenge (PC20 < 12 mg/ml) within last 6 months or demonstrated at visit 1, OR demonstrates improvement in FEV1 of >12% and 200 ml; (3) no history of life threatening asthma; (4) no treatment with bronchial thermoplasty; (5) must be taking inhaled corticosteroid for at least 4 weeks prior to Visit 1		Illumina's Infinium human methylation 450k BeadChip
EVA‐PR study N = 312 atopic N = 171 non atopic	Hispanic Latino	9–20		Population	Atopy: At least one positive specific IgE.	Nasal specimens from above the inferior turbinate (administration of lidocaine 1%)	EWAS‐Illumina's Infinium human methylation 450k BeadChip	8664 CpGs differentially methylated by atopy, significant CpGs related to epithelial barrier function and immune regulation CDHR3, CDH26, SLC9A3, PCSK6, FBXL7, and NTRK1. 1570 CpG‐gene expression pairs were revealed, including 11 of the top 30 EWAS results	Forno et al. (2019) 48
Internal Validation N = 40 atopic asthma N = 40 non atopic non‐asthmatics					Atopic asthma: Parental report of having physician‐diagnosed asthma and at least one episode of wheeze in the previous year, plus at least one positive specific IgE		TWAS‐ RNA seq Illumina NextSeq 500 platform Targeted (15 genes) Pyrosequencing
External Validation (PIAMA) N = 207 atopics N = 255 non atopics	American NonHispanic‐ white	16.4(±0.2) atopics 16.3(±0.2) non atopics	Atopics 55.6% Non atopics 43.6%	N/A	Serum specific IgE to the common aeroallergens.	Nasal brushing from lateral area underneath the right inferior turbinate	Illumina's Infinium human methylation 450k BeadChip
					Sensitization: Specific IgE≥ 0·35 kUA/l. Atopy: Sensitization to any of these allergens. Asthma: Parental “yes” answers to: 1 doctor diagnosis of asthma 2. Wheeze in the last 12 months 3. medication for respiratory or lung problems
547 children: Atopy and controls (ProjectViva)	Multiracial	12.9 (11.9–15.3)	50.6%	Hospital	Current asthma: mother's report of a doctor's diagnosis of asthma since birth reported on the early teen questionnaire plus report of wheeze or asthma medication in the past year at early teen follow‐up.	Nasal swabs from the anterior nares	EWAS‐ Illumina Infinium HumanMethylation450 BeadChips	Overlapping DMRs across phenotypes.More DMRS for FENO • Th2 activation and eosinophilia (EPX, IL4, IL13, PRG2, CLC, ZFMP1) • Solute carriers and intracellular transport genes (SLC25A25, SLC39A4, DNAH17, VTI1A) • T cell activation (LAX) • Oxidative stress (VKORC1L1) • Mucin production (GALNT7). • Gap junction protein gene (GJA4) • 53% replicated CpGs “inner city Consortium” • 61% replicated CpGs “EVA‐PR” studies	Cardenas et al. (2019) 13
					Current allergic rhinitis: mother's report of a doctor's diagnosis of hay fever since birth reported on the early teen questionnaire plus report of sneezing, runny nose, or blocked nose without cold or flu in the past year and current symptoms (moderate‐level nasal congestion/stuffiness, nasal blockage, or trouble breathing through the nose in the past month) at the time of nasal swab.
N = 33 non severe asthma N = 22 severe asthma from exposure Sibling study (ESS) and the genomics of secondhand‐smoke exposure in Pediatric Asthma study (GSEP)	African American (54/55)	Non severe asthma: 12.6 (2.4) Severe asthma 13.7 (3.0)	Non severe asthma: 48.5% Severe asthma: 45.5%	N/A	Asthma severity was defined by symptom frequency using previously validated respiratory symptom score (maximum respiratory symptom score, maxRSS) severe asthma was defined as a symptom score of 3 or 4, while non‐severe asthma was defined as a symptom score of 0, 1 or 2. Allergic children were defined as having a positive doctor's diagnosis of allergy.	Nasal epithelial cell or nasal mucosa sampling with a CytoSoft Brush	Illumina Infinium HumanMethylation450 BeadChip (Illumina) (29 ESS participants) Illumina Infinium MethylationEPIC array (33 GSEP participants)	Differential DNAm were observed between non‐severe and severe. 816 DMPs and 10 DMRs associated with asthma severity. 16 pathways were significantly enriched among the 398 genes associated with these DMPs. 59genes with multiple DMPS that is, LTB4R2, DPP10, IL17RA, CYFIP2, DNAH5, MRPL28, and PTPRN2, TMEM51, WDR25, HIPK3, and KLF11 39 DMP levels associated with mRNA levels	Zhu et al. (2020) 49
Discovery: N = 455 children (PIAMA cohort)	Non Hispanic white	16.3 ± 0.2	47.7%	N/A	Asthma: Presence of at least two‐thirds criteria: (1) doctor‐diagnosed asthma ever, (2) wheeze in the past 12 months, and (3) prescription of asthma medication in the past 12 months	Nasal brushing from lateral area underneath the inferior turbinate with Cytosoft brush after local anesthesia with 1% lidocaine	Infinium HumanMethylation450 BeadChips RNA‐seq‐ Illumina HiSeq2500 platform	81 CpG sites associated with rhinitis 75 were associated with AsRh(mixed asthma/rhinitis phenotype) 8 CpG sites associated with all 3 asthma/rhinitis/asthma and‐or rhinitis including NCF2, NTRK1, GJA4, CYP27B1, and ANO1. 20 CpG site–gene pairs: PCSK6, FBXL7 (F‐box and leucine rich and CISH	Qi et al. (2020) 50
					Rhinitis: Presence of sneezing or a runny or stuffed nose without having a cold in the previous 12 months or the presence of hay fever in the previous 12 months. AsRh: Presence of either asthma or rhinitis
N = 219 asthmatics N = 236 healthy controls (EVA‐PR study)	Hispanic Latino	9–20	N/A	Population	Atopy: 1 positive IgE (≥0.35 IU/ml) to five common allergens in Puerto Rico.	Nasal specimens from above the inferior turbinate (administration of lidocaine 1%)	HumanMethylation450 BeadChips	eQTM enriched in pathways for immune processes and epithelial integrity eQTM methylation‐gene pairs (biologically plausible for lung disease and allergy): PAX8 ECHDC3, LSP1 HLA‐DQB1 FRG1B KANSL1 STAT4, IL32, STAT1, CCR5, CCL5, HLA‐DMB, HLA‐DMA, CXCR6	Kim et al. (2020) 51
					Asthma: physician's diagnosis plus at least one episode of wheeze in the previous year